Methods: OPMNs were prepared by mouth rinse, filtration and Mono-Poly density gradient centrifugation method. The cell death of OPMNs was evaluated by flow cytometry using annexin V-FITC/propidium iodide staining and by trypan blue dye exclusion. One to two millions OPMNs were collected from each of healthy volunteers and more than 90% of freshly prepared OPMNs were viable. The inhibitory effects of antioxidants, caspase and MAP kinase inhibitors were evaluated. Caspase activities were evaluated by substrate cleavage assay. The expression of apoptosis-associated proteins (Bad, phosphorylated-Bad (p-Bad), Bax, Bcl-xL, Bcl-2, cytochrome C) in OPMNs were detected by Western blotting.
Results: OPMNs died via apoptosis and/or necrosis, and its ratio was varied by the types of stimulators. OPMNs died significantly faster at 37 oC than at 4 oC (p<0.05). The cell death of OPMNs was partially inhibited by antioxidants, such as glutathione and N-acetyl-L-cysteine, but not by catalase, SOD, mannitol or vitamin C. Caspase and MAP kinase inhibitors showed little effect. OPMNs showed significantly lower level of caspase-3, -8 and -9 activities, compared to peripheral blood PMNs (PPMNs) (p<0.05). OPMNs expressed higher amounts of unphosphorylated Bad, with weak expression of p-Bad. On the contrary, PPMNs expressed no detectable amount of Bad, with higher amounts of p-Bad. Both OPMNs and PPMNs expressed Bcl-XL, Bax and cytochrome C, but no Bcl-2.
Conclusions: These results suggest that the apoptosis of OPMNs is accelerated by oxidative stress, triggered by mitochondrial dysfunction and possible alterations of Bad expression.